Immunoassay, the primary methodology available for determination of serum digoxin and digitoxin, is limited by interference from endogenous substances of unknown structure and by interference from metabolites, if they are present in serum. The improved analytical method that has been developed, fluorescence derivatization, is specific for unchanged glycoside and the several metabolites and permits testing the following hypotheses: 1) that drug metabolites contribute to immunoassays for serum digoxin or digitoxin as a function of the pathophysiologic state of the patient, and 2) that the endogenous immunoreactive substances which interfere with immunoassays and digoxin in certain pathophysiologic states do not interfere with the newly developed fluorescence derivatization-HPLC method for these drugs and their major metabolites. The specific aims that are designed to test these hypotheses are 1) to investigate potential improvements in the fluorescence derivatization method that has recently been successfully developed for analysis of digoxin and its major metabolites at therapeutic levels in serum, 2) to adapt the fluorescence derivatization method to the analysis of digitoxin and its major metabolites at therapeutic concentrations in serum, 3) to investigate a wide range of pathophysiologic states for interference from endogenous substances or co-administered drugs in the fluorescence derivatization method and in the immunoassays for digoxin and digitoxin, 4) to compare the fluorescence derivatization method with immunoassay for either digoxin or digitoxin in serum samples from patients in the same pathophysiologic states who are at steady-state daily dosing of one of the glycosides and 5) to investigate the possibility of confirming the HPLC identification of metabolites by mass spectrometric analysis. These analyses will be performed on that portion of the serum sample remaining after completion of clinical chemistry tests in selected patients from the large patient population of Ohio State University Hospitals. In carrying out these specific aims, the applicability of the new analytical method for scientific studies of digoxin and digitoxin metabolism and for therapeutic drug monitoring of these glycosides will be evaluated. Results will indicate which disease states, if any, alter metabolism sufficiently to warrant a future in-depth study of digitalis metabolism in that particular pathophysiologic condition.